7/23/2018

Metabolism and the extracellular flux bioanalyzer

Jianhua Zhang Department of Pathology University of Alabama at Birmingham

2018

Bioenergetics and Metabolism

Neurodegeneration: Parkinson’s, Huntington’s, Alzheimers

Cardiovascular disease:

hepatic failure

diabetes mellitus autoimmunity GVHD Renal disease:

proximal tubulopathy

Cancer

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Outline

• how parameters of mitochondrial bioenergetics can be measured • integrate with biology • integrate with metabolomics

The Mitochondrial Electron Transport Chain

+ + H+ H H H+

Q

O2 H2O H+ e‐ H+ ADP + Pi ATP ‐ e H+ H+ Complex I Complex II Complex III Complex IV Complex V (NADH (Succinate (Ubiquinol‐ (Cytochrome (ATP Dehydrogenase) dehydrogenase) cytochrome c c oxidase) synthase) How can we measure mitochondrial oxidoreductase) function?Inhibitors Rotenone TTFA Myxothiazol Cyanide Oligomycin Antimycin A NO˙

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Measuring Mitochondrial Function

Extracellular Oxygen Acidification Rate Consumption Rate (ECAR) (OCR) = = Glycolysis Mitochondrial Respiration

Measuring Mitochondrial Function in Cells

PMP Succ Oligo FCCP Ant-A Rot ADP

FCCP H+ 200 HV X 180 160 + 140 O2 H H2O ADP ATPH+ 120 100 X 80

OCR (pmol/min) 60 H+ 40 CII-FCCP St 3 St 4 20 CII-ADP State 3 0 RCR = 0 20406080100 State 4 Time (min)

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Letting the cell drive its own mitochondria

Cell fuel Antimycin A 500 H+ X FCCP 400 + FCCP O2 H H2O ADP ATPH+ /min) 2 300 X Oligomycin RESERVE CAPACITY H+ MAXIMAL 200

OCR (pmol O (pmol OCR ATP MAXIMAL 100 BASAL LEAK NON-MITOCHONDRIAL 0

Time

Measuring Mitochondrial Function in Cells

350

300 Platelets FCCP 250 200

150

100

OCR (pmol/min) OCR 50

0 CI-F CIV-F CII-F CII-ADP

7 RCR (CII) 6 160 Mono 140 5 120 4 100 80 3 60 2 40 20 4 State3/state 1 OCR (pmol/min) OCR 0 0 CI-F CIV-F CII-F CII-ADP Platelets Mono

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Comparing Mitochondrial Activity to Cellular Bioenergetics

Mitochondria Cellular Bioenergetics 160 160 Mono F 140 140 AA 120 120 100 100 O 80 80 Mono 60 60 40 40 Maximal OCR (pmol/min)

OCR (pmol/min) Basal 20 20 Non-Mito 0 0 CI-F CII-F CIV-F 0 20406080 Time (min)

350 Platelets 350

300 300

250 250 F AA 200 200 O Platelets 150 150

100 100 OCR (pmol/min)

50 OCR (pmol/min) 50 Basal Maximal Non-Mito 0 0 CI-F CII-F CIV-F 0 20406080 Time (min)

The energy profile represents a fundamental bioenergetic program

Cardiomyocytes Hepatocytes Endothelial Cells 350 A+R 100 40 A+R A 300 80 30 250 F F F O 200 60 O 20 150 O 40 /min/μg protein) /min/μg protein) /min/μg protein) 2 2 100 2 10 20 50 (pmol O (pmol O (pmol O 0 0 0 Oxygen Consumption Rate Oxygen Consumption Rate Oxygen Consumption Rate 0 10203040 0 204060 0 204060 Time (min) Time (min) Time (min)

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Differentiation changes cellular bioenergetics

OCR: Oxygen consumption rate Schneider et al FRBM 2011

Inhibition of mitochondrial function induces compensatory upregulation of glycolysis

H+ H+ IMS

Matrix O2 H2O ADP ATP H+

18 160 FCCP Antimycin 16 140 14 Rotenone Oligomycin 12 120 10 100 8 80 Control 6 0.1nM from control) 4 60 2 OCR (% of Basal) 40 0.5nM Δ ECAR (fold change 0 10nM 20 50nM 0 0 20406080100 Time (min) rotenone

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Effects of NO on OCR during hypoxia-reoxygenation

Cortical Neurons Reoxygenation

20 180 Control DetaNO 250 µM DetaNONOate (DetaNO) 18 Series5 160 16 250μM 140 14 120 O

12 2

100 (mmHg) 10 80 8 60 6 Hypoxia Chamber (1%) Vacuum Argon OCR (pmol/min/µg ptn) 40 4

XF24 Controller 20 Vacuum 2 Chamber 0 0 0 20 40 60 80 100 120 140 160 180 200 220 240 260

Time (min) Gloves Benavides et al 2013

Pathological fibrosis Metabolic Plasticity in Lung

myofibroblasts extracellular matrix tissue deformation Normal lung Fibrotic lung (bleomycin)

objective 10 objective 10

Bernard et al JBC 2015

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Metabolic Switch In Platelets

Data represented as mean±SEM. n=3. One-way ANOVA, Post-hoc tukey test. * p<0.01 different from Ctrl # p<0.01 different from Thrombin Ravi, S et.al. 2015. Plos One. 10(4):e0123597

Integration with Metabolomics

Thrombin

Slatter et al Cell Metabolism 2016

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Mitochondrial Quality Control and Autophagy

Lysosome

3MA GABARAPL1 CQ Baf

LC3I PE LC3‐II

LC3I LC3‐II

Initiation Elongation Fusion

Downregulation of autophagy reprograms redox tone and bioenergetic health in cancer cells

GABARAPL1 KO

Autophagy  flux  Lysosome number  LAMP1

 VDAC1

 Mitochondria  TMRM number  MitoTracker

 HNE‐protein  Basal OCR adducts  ROS ?

 GSH  ATP  mtDNA damage  Resistance to HNE‐  Proliferation induced cell death Boyer‐Guittaut et al 2014

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Autophagy inhibition suppresses complexes I and II activities in permeabilized primary neurons

250 ADP Control P/M Suc 200 Baf 10 PMP R ADP A CQ 40 150

100

50 OCR (pmol/min)

0 0 20406080100 Time (Minutes)

150 200 160 100 120 * * * 50 * 80 40

OCR (pmol/min) 0 OCR (pmol/min) 0 Complex I Complex II Control Baf 10 CQ 40 Control Baf 10 CQ 40

Redmann et al Redox Biology 2017

Autophagy inhibition suppresses mitochondrial function in primary neurons

100 Control Baf 10 80 CQ 40 O F A 60

40 50 20

OCR * * OCR (pmol/min/µg protein) 0 0 0 20406080 Reserve Capacity Time (Minutes) Control Baf 10 CQ 40

40 40 100 * 20 * 20 * 50

OCR * OCR *

OCR * 0 0 0 Basal ATP‐Linked Maximal Control Baf 10 CQ 40 Control Baf 10 CQ 40 Control Baf 10 CQ 40

Redmann et al Redox Biology 2017

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Comprehensive Mitochondrial Assessment

Autophagy LC3‐II Western blot Imaging LC3 puncta Mitochondria Assessing morphology Imaging of Fragmentation autophagy Mitophagy Imaging MT/LT colocalization and mitophagy Mitochondrial Mitochondrial mass ‐ protein levels Network Mitochondrial mass ‐ mtDNA copy

mtDNA and protein Damage

TCA Assessing Metabolomics Glycolysis metabolism Mass spectrometry Glutaminolysis Assessing mitochondrial Bioenergetic Intact cells bioenergetic Assessment Permeabilized cells function Seahorse XF analyzer

Redmann et al Redox Biology 2018

Assessment of autophagy

Control Baf 10nM CQ 40µM 1.5 5 * * LC3‐I 1 4 3 LC3‐II 0.5 2

LC3‐I/Actin 1 LC3‐II/Actin Actin 0 0 Control Baf 10 CQ 40 Control Baf 10 CQ 40

Redmann et al. Redox Biology 2017

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No change of mitochondrial mass but significant increase of mitochondrial DNA damage in response to autophagy inhibition

1.4 3.5 * 1.2 3 1 2.5 0.8 2 * 0.6 1.5

mtDNA/nDNA 0.4 1 per 16Kb DNA Lesion Frequency 0.2 0.5 0 0 Ctrl Baf CQ Con Baf CQ ‐0.5

Redmann et al. Redox Biology 2017

Integration with Metabolomics

1 Pyruvate 2 Citrate 0.1 cis‐Acon * *# *# * 0 Pyruvate 0 Con Baf CQ Con Baf CQ 0.0 Acetyl‐CoA Con Baf CQ Citrate 0.5 Malate 0.1 Isocitrate * Cis‐Aconitate * * Oxaloacetate Isocitrate 0.0 0.0 Con Baf CQ All Con Baf CQ Malate measurements Oxalosuccinate in µg/mL α‐Ketoglutarate 0.5 AKG Fumarate *# 0.2 Fumarate * * Succinyl‐CoA 0.0 Succinate Con Baf CQ 0.0 20 Con Baf CQ 1 Succinate Glutamate * * 0 0 CS Activity Con Baf CQ 15 Con Baf CQ Glutamine * * 1 10 * * 5 p<0.05 *To control #Between Baf and CQ 0 0 nmol/min/mg Con Baf CQ Redmann et al Redox Biology 2017 Con Baf CQ

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Screening for Drugs

From Human Blood; 4 Bioenergetic Programs

500xg 700xg Antibody

15 min 30 min Incubation Buffy coat diluted 1:4

MNC PRP 1.077 PMN Buffy Coat 1.119 RBC

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Energy mapping of leukocyte and platelets

10 Highly Energetic 9 Oxidative 8

7 Plt

6 Mono 5 Lymph 4 3 Neutro 2 Low Glycolytic OCR (pmoles/min/µg protein) 1 Energy 0 0123456 ECAR (mpH/min/µg protein)

• PBMCs are a mixed population with different metabolic programs

Mitochondrial defect in patients

Bioenergetic Parameters 38 year old Male 12.0 Episodes of 10.0 Healthy Mitochondrial Disease extreme fatigue 8.0 # 6.0 # 4.0 2.0 Monocytes OCR(pMol/min/10,000 cells) 0.0 Basal ATP‐Linked Maximal Reserve Capacity

Mitochondrial Respiratory Complexes Bioenergetic Health Index 7 Healthy 80 6 Mito Disease 70

5 60 50 4 # 40 # 3 30 2 20 OCR(pMol/min/10,000 cells) 1 Bioenergetic Health Index 10

0 0 Complex ‐I Complex‐IV Healthy Mitochondrial Disease

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Monocyte energetics vs skeletal muscle (vastus lateralis)

Skeletal Muscle Skeletal Muscle Fibers Mitochondria

Anthony Molina: Wake Forest School of Medicine Redox Biology, 2016

Acknowledgment

Samantha Giordano Qiuli Liang Matt Dodson Matt Redmann Israr Ahmad Willayat Wani Xiaosen Ouyang David Westbrook

UAB Targeted Metabolomics Balu Chacko & Proteomics Laboratory Stephen Barnes Taylor Berryhill

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